Neural cell adhesion molecule (NCAM) association with PKCβ2 via βI spectrin is implicated in NCAM-mediated neurite outgrowth

Abstract

In hippocampal neurons and transfected CHO cells, neural cell adhesion molecule (NCAM) 120, NCAM140, and NCAM180 form Triton X-100–insoluble complexes with βI spectrin. Heteromeric spectrin (αIβI) binds to the intracellular domain of NCAM180, and isolated spectrin subunits bind to both NCAM180 and NCAM140, as does the βI spectrin fragment encompassing second and third spectrin repeats (βI2–3). In NCAM120-transfected cells, βI spectrin is detectable predominantly in lipid rafts. Treatment of cells with methyl-β-cyclodextrin disrupts the NCAM120–spectrin complex, implicating lipid rafts as a platform linking NCAM120 and spectrin. NCAM140/NCAM180–βI spectrin complexes do not depend on raft integrity and are located both in rafts and raft-free membrane domains. PKCβ2 forms detergent-insoluble complexes with NCAM140/NCAM180 and spectrin. Activation of NCAM enhances the formation of NCAM140/NCAM180–spectrin–PKCβ2 complexes and results in their redistribution to lipid rafts. The complex is disrupted by the expression of dominant-negative βI2–3, which impairs binding of spectrin to NCAM, implicating spectrin as the bridge between PKCβ2 and NCAM140 or NCAM180. Redistribution of PKCβ2 to NCAM–spectrin complexes is also blocked by a specific fibroblast growth factor receptor inhibitor. Furthermore, transfection with βI2–3 inhibits NCAM-induced neurite outgrowth, showing that formation of the NCAM–spectrin–PKCβ2 complex is necessary for NCAM-mediated neurite outgrowth.

Immobilized Pool of NCAM180 in the Postsynaptic Membrane Is Homeostatically Replenished by the Flux of NCAM180 from Extrasynaptic Regions

“…In agreement with this idea, synaptic activity is accompanied by cell surface delivery of NCAM from intracellular organelles (52) and may therefore increase its homophilic interactions. Clustering of NCAM induces the assembly of the spectrin meshwork (25,35), which in turn may promote the retention of NCAM180 at activated synapses. Furthermore, although the interaction of NCAM180 with spectrin is not Ca 2ϩ -dependent (53), synaptic activity-induced Ca 2ϩ influx induces redistribution to and immobilization in synapses of CaMKII␣ (54 -56), which binds to the NCAM-spectrin complex (25) and may thus also increase the interactions between NCAM and the cytoskeleton.…”

“…Binding to synaptic scaffold proteins is often considered as a mechanism for the recruitment of proteins to synapses (47). The intracellular domain of NCAM directly interacts with the spectrin scaffold accumulating in synapses (25,35). The extracellular domain of NCAM binds to the extracellular domains of NCAM molecules on adjacent membranes, thereby mediating homophilic cell adhesion interactions (48).…”

“…To analyze this possibility, we developed a mathematical model to predict the behavior of NCAM180GFP in the membrane. NCAM180 has been shown to bind via the intracellular domain to spectrin molecules (25,35) and is expected to interact via the extracellular domain with extracellular domains of other adhesion molecules in the synaptic cleft (Fig. 3A).…”

“…First, we performed FRAP experiments in which this interaction was disrupted by co-transfecting wild-type hippocampal neurons with the spectrin fragment ␤I 2-3 containing the NCAM binding site and disrupting the association between NCAM180 and endogenous spectrin (35). For comparison, we analyzed neurons co-transfected with the ␤I 3-5 spectrin fragment, which does not interact with the intracellular domain of NCAM180 (35). The preferential accumulation of NCAM180GFP in the postsynaptic membrane was reduced in neurons transfected with the ␤I 2-3 spectrin fragment (compare prebleach levels in Fig.…”

Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation

“…NCAM has previously been found to localize and act in membrane rafts in C2C12 myoblasts as well as neuronal cells and various NCAM transfected cell lines [50][51][52]. To investigate the localization of NCAM in relation to known membrane raft markers in primary satellite cells, satellite cell cultures amplified for 96 hours after harvest from the mouse were either fixed (as a proliferating sample) or grown for an additional 48 hours under differentiating conditions then fixed.…”